Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China

Journal Pre-proof Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China Wenchao Sun, Li Wang, Haixin Huang, Wei Wang, Liang Cao, Jinyong Zhang, Min Zheng, Huijun Lu

PII:

S0168-1702(19)30627-6

DOI:

https://doi.org/10.1016/j.virusres.2020.197869

Reference:

VIRUS 197869

To appear in:

Virus Research

Received Date:

1 September 2019

Revised Date:

17 January 2020

Accepted Date:

17 January 2020

Please cite this article as: Sun W, Wang L, Huang H, Wang W, Cao L, Zhang J, Zheng M, Lu H, Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China, Virus Research (2020), doi: https://doi.org/10.1016/j.virusres.2020.197869

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China

Wenchao Suna,1, Li Wangb 1, Haixin Huanga 1, Wei Wangc, Liang Caoc, Jinyong Zhangc, Min Zhengd, Huijun Lua*

Institute of Virology, Wenzhou University, Wenzhou 325035, China

b

Shandong New Hope Liuhe Group Company, Qingdao 266100, China

c

ro of

a

Institute of Military Veterinary Medicine, The Academy of Military Medical

d

re

Guangxi Centre for Animal Disease Control and Prevention, Nanning 530001, China

1These

lP

authors contributed equally to this work. Corresponding Author, Huijun Lu Emails: [email protected] (HL)

The China lineage strains have become predominant in the Shandong Province.

Jo



ur

Highlights

na

*

-p

Sciences, Changchun 130122, China



Five strains contained the same two discontinuous deletions as the Vietnam/Laos/Thailand lineage isolates in the Nsp2 and Nsp3 regions.



Three strains with a novel pattern of deletions were observed for the first time in the Nsp2 and Nsp3 regions.

1

Abstract Porcine deltacoronavirus (PDCoV) is the etiological agent of acute diarrhoea and vomiting in pigs, threatening the swine industry worldwide. Although several PDCoV studies have been conducted in China, more sequence information is needed to understand the molecular characterization of PDCoV. In this study, the partial ORF1a,

ro of

spike protein (S) and nucleocapsid protein (N) were sequenced from Shandong

Province between 2017 and 2018. The sequencing results for the S protein from 10 PDCoV strains showed 96.7%-99.7% nucleotide sequence identity with the China

-p

lineage strains, while sharing a lower level of nucleotide sequence identity, ranging

re

from 95.7-96.8%, with the Vietnam/Laos/Thailand lineage strains. N protein sequencing analysis showed that these strains showed nucleotide homologies of 97.3%

lP

to 99.3% with the reference strains. Phylogenetic analyses based on S protein

na

sequences showed that these PDCoV strains were classified into the China lineage. The discontinuous 2+3 aa deletions at 400-401 and 758-760 were found in the Nsp2

ur

and Nsp3 coding region in five strains, respectively, with similar deletions having been identified in Vietnam, Thailand, and Laos. Three novel patterns of deletion were

Jo

observed for the first time in the Nsp2 and Nsp3 regions. Importantly, those findings suggest that PDCoV may have undergone a high degree of variation since PDCoV was first detected in China. Keywords: Porcine deltacoronavirus (PDCoV), Spike protein, Nucleocapsid protein, ORF1a, Deletion 2

1. Introduction Porcine deltacoronavirus (PDCoV) was first discovered in 2009 in Hong Kong from swine fecal samples (Woo et al., 2012). PDCoV was recognized as the causative agent of acute diarrhoea and vomiting in pigs (Hu et al., 2015; Song et al., 2015; Wang et al., 2014). Since its appearance, PDCoV has caused significant economic

ro of

losses for the swine industry worldwide. PDCoV is a member of the genus

Deltacoronavirus in the family Coronaviridae and is an enveloped virus that has a

positive-sense single-stranded RNA (+ssRNA) genome of 25.4 kb in length (Lee and

-p

Lee, 2015; Phan et al., 2018). The PDCoV genome has seven major open reading

re

frames (ORFs). Two overlapping ORFs (ORF1a and ORF1b) encode two replication-associated proteins, which are both autoproteolytically cleaved into 15

lP

nonstructural proteins (Nsp2 to Nsp16) (Wang et al., 2018a; Woo et al., 2010). The

na

remaining ORF encodes the spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). Additionally, three accessory proteins were

ur

identified: nonstructural protein 6 (NS6), NS7, and NS7a (Fang et al., 2017; Fang et al., 2016; Luo et al., 2016).

Jo

The S protein is the most variable protein among the PDCoV genes, with only

96.0%–100% amino acid sequence identity between Chinese and American strains (Zhang et al., 2019b). The S protein plays a pivotal role in the viral entry and stimulates the induction of neutralizing antibodies in the natural host (Chen et al., 2019c; Chiou et al., 2017; Lin et al., 2016; Zhang et al., 2017). N protein is a 3

conservative target for virological detection by PCR (Lee and Lee, 2015). N protein also plays an important role in viral pathogenesis (Chen et al., 2019b; Likai et al., 2019; Shi et al., 2017; Zhang et al., 2015; Zhang et al., 2014). PEDV N protein can antagonize beta interferon and interferon-λ production (Ding et al., 2014; Shan et al., 2018). SARS-CoV N protein can bind to DNA in vitro (Chen et al., 2007). hCoV-OC43 N protein interacts with the transcription factor nuclear factor-kappa B

ro of

(NF-κB) (de Haan and Rottier, 2005). The ORF1a region is the most variable region of the PDCoV genome and substitutions, deletions and insertions have been observed

in the Nsp2 and Nsp3 coding region in Vietnam, Thailand, and Laos (Lorsirigool et al.,

-p

2017; Wang et al., 2015).

re

To determine the molecular epidemiology and genetic variations of PDCoV in China, the partial ORF1a, S protein and N protein genes of 10 PDCoV strains from

lP

different pig farms located in Shandong Province were sequenced and analysed. This

na

study may provide valuable information for the molecular epidemiology of PDCoV

ur

and its emerging variants in China.

2. Materials and methods

Jo

2.1 Sample collection

To monitor the prevalence and sequence properties of PDCoV in Shandong

Province, China, a total of 58 porcine samples, including 21 faecal samples and 37 intestinal samples, were collected from different commercial swine from September 2017 to December 2018. All samples were stored at -80°C and were subsequently 4

used for RNA extraction. 2.2 RNA extraction and PDCoV detection

RNAs were extracted from the samples using the RNAeasy mini kit (TaKaRa BIO INC., Dalian, China) according to the manufacturer’s instructions. To detect, differentiate and sequence PDCoV, the One Step RT-PCR kit (TaKaRa Co., Dalian, China) was used to synthesis cDNA.

ro of

Ten PDCoV-positive samples were selected for the partial ORF1a, S protein and

N protein sequences. The primer sets are listed in Table 1. TGEV, PEDV and PoRV were detected as described previously (Wang et al., 2018c). The PCR conditions were

-p

as follows: an initial PCR activation temperature of 95 °C for 5 min, followed by 35

re

cycles of denaturation at 94 °C for 30 s, annealing at 55 °C~65 °C for 50 s, and extension at 72 °C for 120 s and another extension at 72 °C for 10 min. The PCR

lP

products were ligated into the pMD18-T cloning vector (TaKaRa Co., Dalian, China)

na

and sequenced.

2.3 Nucleotide sequencing and phylogenetic analysis

ur

The partial ORF1a, S protein and N protein sequences of PDCoV were independently used for sequence alignments and phylogenetic analyses. The

Jo

nucleotide and deduced amino acid sequences were assessed using BioEdit 7.0. All the sequences were aligned using the MEGA5 program, and phylogenetic trees were constructed using the neighbour-joining method. The reliability of the branching orders was evaluated by the bootstrap test (n =1,000). After nucleotide homology comparison and screening, 29 representative strains were selected from 102 strains for 5

molecular evolutionary analyses (Table 2 and Table S1). In addition, these strain sequences have been previously published as reference sequences (Suzuki et al., 2018; Zhang et al., 2019a; Zhang et al., 2019c; Zhang et al., 2019d).

3. Results 3.1 Sample screening and genome sequencing

Twelve of fifty-eight (20.68%) field samples were positive for PDCoV, while the

ro of

PEDV infection rate was 34.48% (20/58), and the co-infection rate of PEDV and

PDCoV was up to 50.00% (10/20). 12 PDCoV positive samples were identified from 4 faecal samples and 8 intestinal samples. PRV was identified in 5 of 58 samples.

-p

Two of fifty-eight samples were found to be positive for TGEV. PRV/PEDV and

re

TGEV/PEDV co-infections were 15.00% (3/20) and 5.00% (1/20), respectively. None of the PDCoV-positive samples were positive for TGEV and PRV. PDCoV/PEDV

lP

co-infections were the most common.

na

To obtain the sequence, the complete S protein, N protein and partial ORF1a genes were amplified in 10 PDCoV-positive samples (Table 1). The 10 strains were named SD03-2018,

SD05-2018,

SD06-2018,

SD07-2018,

SD08-2018,

ur

SD01-2018,

SD09-2018, SD10-2018, SD11-2018, and SD12-2018. The GenBank accession

Jo

numbers of the complete S protein, N protein and partial ORF1a genes were as follows:

MN173803-MN173812,

MN173783-MN173792,

and

MN173802, respectively. 3.2 Phylogenetic analysis of the S, N and partial ORF1a/1b genes 3.2.1. Phylogenetic analysis of S protein 6

MN173793-

Phylogenetic analysis of the nucleotide sequences of the S protein sequences indicated that all strains worldwide can be categorized into three lineages: the China lineage, USA/Japan/South Korea lineage and Vietnam/Laos/Thailand lineage. All new PDCoVs strains from Shandong in our study belonged to the China lineage (Figure 1A). The sequence alignment results showed that these new strains shared nucleotide

ro of

sequence homologies of 97.5%-99.9% with each other. They also shared up to 96.7%-99.7% nucleotide sequence similarity with the representative China lineage

strains (HKU15-44, CHN-AH-2004, CHN-HN-2014 and CHN-HG-2017) and

-p

97.5%-98.6% with the representative USA/Japan/South Korea lineage strains

re

(OhioCVM1/2014, IL/2014/026PDV_P11, YMG/JPN/2014 and KNU14-04). By contrast, the new strains showed lower 95.7-96.8% nucleotide sequence identity with

lP

the representative Vietnam/Laos/Thailand lineage strains (Vietnam/Binh21/2015,

na

Thailand/S5011/2015 and P1_16_BTL_0115 /2016/Lao). 3.2.2. Phylogenetic analysis of N protein

ur

A phylogenetic tree was constructed using the N protein sequences from the strains isolated from Shandong and the references strains (Figure 1B). SD07-2018,

Jo

SD11-2018 and SD12-2018 with CHN-HG-2017 were classified in a group. Seven strains and HNZK-02 and CH/JXJGGS01/2016 were classified in a branch. The deduced amino acid identity values of N protein sequences were analysed, and shared nucleotide sequence homologies of 98.4%-99.9% were found among 10 strains. They shared 97.9-99.3%, 98.3%-98.9%, and 97.3%-98.9% sequence identity with the 7

China

lineage

strains,

USA/Japan/South

Korea

lineage

strains

and

Viet

Nam/Laos/Thailand lineage strains, respectively. As shown in Figure 2, all the PDCoV N protein sequences were the same length (1029 nt) and encoded 342 amino acid residues. Compared to other structural proteins, N protein is the most conserved structural proteins and remains the primary target protein for current diagnostic tests (McBride et al., 2014). Recently, a recombinant N indirect

enzyme-linked

immunosorbent

assay

(ELISA)

ro of

protein-based

(rPDCoV-N-ELISA) was established to detect PDCoV IgG antibodies (Su et al.,

2016). Similar ELISA test has been done in Taiwan (Hsu et al., 2018). Compared with

-p

the reference strain HKU15-44, the following aa changes were detected in

re

CHN-AH-2004, SD, P1_BTL_0115/2016/Lao and Thailand/S5015L/2015 at 43V/A, 163S/P, and 265I/M, respectively. Several amino acid mutations were detected in 10

lP

strains at positions 18L/P, 67Y/T, 82G/S, 215G/A/F, 216S/D, 218D/G, 222G/A,

na

223M/V, 291P/S, 310P/V, and 329E/V. N protein is a multifunctional protein, the key conserved amino acid site mutations affect their function, for example, the Arg-76 and

ur

Tyr-94 residues in the IBV N protein are critical for RNA binding, SARS-CoV N protein also exists Arg-94 and Tyr-122 residues (Tan et al., 2006). PDCOV is difficult

Jo

to isolate and there are few reverse genetic studies. So far, there have been no related reports that mutations in key amino acids of the N protein will affect the current diagnostic tests. 3.2.3. Phylogenetic analysis of the partial ORF1a gene A phylogenetic tree was generated based on the deduced aa sequence of the partial 8

ORF1 gene. SD01-2018, SD03-2018, SD05-2018, and SD08-2018 strains were placed into one subgroup with the Vietnam/Laos/Thailand lineage strain and CH/Sichuan/S27/2012 strain. The other subgroup containing SD07-2018, SD09-2018, SD10-2018 and SD11-2018 was closer to the Chinese strain CHN-HG-2017. The SD12-2018 strain was placed into one subgroup (Figure 1C). The partial ORF1a gene of the 10 strains shared 95.9%-98.7% nucleotide identity

ro of

with the China lineage strains (HKU15-44, CHN-AH-2004, CHN-HN-2014 and CHN-HG-2017). These strains shared 96.0%–97.5% nucleotide identity with the

USA/Japan/South Korea lineage strains (OhioCVM1/2014, IL/2014/026PDV_P11,

-p

YMG/JPN/2014 and KNU14-04) and 95.3%–97.9% nucleotide identity with the

and P1_16_BTL_0115 /2016/Lao).

re

Vietnam/Laos/Thailand lineage strains (Vietnam/Binh21/2015, Thailand/S5011/2015

lP

3.3 Amino acid variants in the spike proteins

na

Compared with the USA/Japan/South Korea lineage and Vietnam/Laos/Thailand lineage strains, the China lineage strains have 3-nt TAA deletions (52N), leading to

ur

the lack of an asparagine in the S gene. This may be the most important feature of the Chinese lineage strains, except for CH/Jiangsu/2014 (Zhang et al., 2019d). Sequence

Jo

analysis showed that 27 aa mutations were detected in S1 (Figure 3). PDCoV employs host aminopeptidase N (APN) as an entry receptor and interacts with APN via domain B of its S1 protein (residues 298-425) (Li et al., 2018b; Shang et al., 2018). The S1 protein region mutation (residues 229, 231, 234, 276, 348 and 350) may have an impact

on

virus

entry

or

recognition 9

by

neutralizing

antibodies.The

Vietnam/Laos/Thailand lineage strains had the following unique substitutions: 14V/A, 42S/D, 99H/Q,107H/Q, 149H/AR, 159E/D, 178E/Q, 229H/Q, 348M/L and 350T/R. The USA/Japan/South Korea lineage strains only had the unique substitutions 23F/L, 234N/RS, 551A/V, 670L/I, 698A/S, 907P/S and 1016V/I. Among these 4 strains, SD07-2018, SD09-2018, SD11-2018 and SD12-2018 had another 2 unique substitutions at positions 513F/S and 534C/N.

ro of

3.4 Novel amino acid deletions or insertions were detected in the partial ORF1a gene

The partial ORF1a gene has the highest genetic diversity in the genomes of the

-p

PDCoV strains (Xu et al., 2018). To determine whether the strains in this study

re

possessed the characteristic deletions in the ORF1a gene found in the Thailand PDCoVs previously described, the partial ORF1a sequences containing the Nsp2 and

lP

Nsp3 hypervariable region (HVR) from 10 PDCoV strains were sequenced and

na

aligned with the reference isolates, especially those with known pathogenicity, OhioCVM1/2014, IL/2014 /026PDV_P11, YMG/JPN/2014, and CHN-HG- 2017.

ur

Compared with the OhioCVM1/2014, IL/2014 /026PDV_P11, and YMG/JPN/2014 strains, five strains (SD01-2018, SD03-2018, SD05-2018, SD06-2018 and

Jo

SD08-2018) in the partial ORF1a gene had the same length of 1255 nucleotides, containing the same two discontinuous deletions of 2+3 amino acids at positions 400-401 and 758-760 as the Vietnam/Binh21/2015 and Thailand/S5011/2015 strains, suggesting that these strains are the dominating strain circulating in Shandong Province. Four strains (SD07-2018, SD09-2018, SD10-2018 and SD11-2018) in the 10

partial ORF1a gene had a length of 1,249 nucleotides, containing a continuous 0+7 amino acid deletion at position 755-761. The SD12-2018 strain in the Nsp3 gene had a length of 1,261 nucleotides, and a 0+3 amino acid deletion at position 758-760. By contrast, no deletions or insertions were detected within the ORF1a gene in the Chinese strains collected early in the study. Additionally, the SD strain contained a 2+0 deletion at positions 400-401 in the Nsp2 gene.

ro of

As shown in Figure 4, five strains (SD01-2018, SD03-2018, SD05-2018,

SD06-2018 and SD08-2018) with a 7-aa deletion in ORF1 regions had substitutions identical with those of the Thailand/S5015L/2015 strain at positions 397A/S, 427I/L,

-p

447A/T, 547T/I, 550N/D, 554T/A, 599F/L, 647A/V, 670P/L, 717D/N, and 762K/E.

re

Additionally, five strains had unique substitutions at positions 529E/D, 778P/S and 780P/I. Notably, four strains (SD07-2018, SD09-2018, SD11-2018 and SD12-2018)

na

lP

carried substitutions at position 401 L/M.

4. Discussion

ur

Several enteric coronaviruses have been identified from diarrhoeic piglets, such as porcine epidemic diarrhoea virus (PEDV) (Li et al., 2012), transmissible

Jo

gastroenteritis virus (TGEV) (Xia et al., 2018), Swine acute diarrhea syndrome coronavirus (SADS-CoV) , also named as Swine enteric alphacoronavirus (SeACoV) (Fu et al., 2018; Zhou et al., 2018) and PDCoV. Outbreaks of these enteric coronaviruses have been reported in China, causing substantial economic losses in the swine industry (Qing et al., 2016; Zeng et al., 2015; Zhou et al., 2019). In 2014, 11

PDCoV was first detected in China, causing tremendous financial losses in the swine industry, and the virus has rapidly spread nationwide (Dong et al., 2015; Liu et al., 2018; Mai et al., 2018a; Song et al., 2015; Wang et al., 2018b). Thus, the molecular characterization of these PDCoVs is a major focus of Chinese virological research. Although several studies have shown an obvious increase in the genomic diversity of PDCoV in China (Dong et al., 2016; Liang et al., 2019; Mai et al., 2018b), previous

PDCoVs

based

on

the

ORF1

gene

are

not

ro of

studies have focused mainly on the S genes, while the genetics of the Chinese well

characterized.

The

Vietnam/Laos/Thailand lineage strains had 6-nt (AGTTTG) and 9-nt (GAGCCAGTC)

-p

deletions in ORF1a (Le et al., 2018). In 2015, only a few strains with similar deletions

re

have been reported in China, such as the CH/Sichuan/S27/2012 strain with the 6-nt and 9-nt deletions in the ORF1a (Wang et al., 2015). The ORF1a deletion region

lP

encoded Nsp2 and Nsp3 proteins. The Nsp3 protein acts as a scaffold protein to

na

interact with itself and bind other viral Nsps or host proteins (Nogales et al., 2012; Yuan et al., 2015). Nsp3 protein contains one or two papain-like protease (PLpro)

ur

domain(s) with deubiquitinating (DUB) and deISGylating activities in SARS-CoV and MERS-CoV (Alfuwaires et al., 2017; Neuman, 2016). In MHV, the Nsp3 protein

Jo

ubiquitin-like domains could interfere with pathways involving ubiquitinylated or ISGylated host targets, thereby leading to the disruption of host anti-viral signal transduction or protein degradation (Chen and Makino, 2004). Nsp3 protein was also recommended as a marker for monitoring coronavirus evolution and for surveying the molecular epidemiology in lineage C betaCoVs (Forni et al., 2016). Additionally, the 12

MERS-CoV Nsp3 Arg911Cys mutation is an example of adaptive evolution(Shokri et al., 2019). In TGEV Nsps 2, 3, and 8 were incorporated into the CoV virions, involving in CoV replication (Nogales et al., 2012). Mutation and recombination are important mechanisms for PDCoV evolution. The USA/Japan/South Korea lineage strains were characterized by no discontinuous deletions in the Nsp2 and Nsp3 coding regions, while the Vietnam/Laos/Thailand

ro of

lineage strains showed a discontinuous 5-aa deletion (2aa+3aa) at 400-401 and

758-760 in the Nsp2 and Nsp3 coding regions. These regional deletions were also

observed in a Chinese CH/Sichuan/S27/2012 strain. Here, we found 5 novel strains

-p

with the same deletion pattern of “2+3aa” in the Nsp2 and Nsp3 coding regions.

re

Interestingly, the SD12-2018 and CHN-HG-2017 strains have a continuous “0+3aa” in the Nsp3 deletion at 758-760. Additionally, SD07-2018, SD09-2018, SD10-2018

lP

and SD11-2018 strains have a continuous “0+7aa” deletion at 755-761 in Nsp3. Two

na

continuous aa deletion in Nsp2 was also identified in a Chinese strain, SD, which has two“2+0aa” deletions at position 400-401. Remarkably, natural deletions and

ur

insertions occurred in the ORF1a sequence, and these have led to genome size differences among PDCoV strains. However, the role of double deletions in the ORF1

Jo

of this virus remains unclear. Thus, the current results indicate that the novel deletion of Nsp2 and Nsp3 may provide another approach to the diagnosis. The PDCoV S protein is a glycoprotein of approximately 1,383 amino acids (aa) with an apparent molecular mass of 180 kDa. Studies of genomic sequences analyses using S protein genes have shown that PDCoV can be further divided into three 13

lineages:

the

China

lineage,

USA/Japan/South

Korea

lineage

and

Vietnam/Laos/Thailand lineage (Zhang et al., 2019d). The Vietnam/Laos/Thailand lineage has been detected in Southeast Asia (Janetanakit et al., 2016; Lorsirigool et al., 2017; Lorsirigool et al., 2016; Saeng-Chuto et al., 2017). The USA/Japan/South Korea lineage has a worldwide distribution (Jang et al., 2017; Nelson et al., 2019; Niederwerder and Hesse, 2018; Perez-Rivera et al., 2019; Suzuki et al., 2018). The

ro of

China lineage has been the predominant lineage in China since 2014 (Li et al., 2018a;

Zhang et al., 2019a). S protein-based phylogenetic trees showed that 7 strains

belonging to the China lineage were classified into a minor branch with

-p

CHN-HG-2017. The evidence showed that the PDCoV strains of China have genetic

re

diversity.

The PDCoV spike (S) protein comprises S1, a receptor-binding subunit, and S2, a

lP

membrane fusion subunit. The S1 domain is important for recognizing and binding to

na

cell receptors. Additionally, the S1 domain contains several neutralizing epitopes that stimulate the induction of neutralizing antibodies. The S2 domain is involved in

ur

triggering the fusion of the viral envelope and target cell membrane. Therefore, the S protein has been the primary target for the development of vaccines against PDCoV

Jo

and for determining genetic relatedness among PDCoV isolates. In this study, compared with the USA/Japan/South Korea lineage and Vietnam/Laos/Thailand lineage strains, the 10 strains showed one amino acid (52N) deletion in the S protein. A comparison of the antigenic index profiles of the S protein among the IL/2014/026PDV_P11, CHN-HG-2017, and Vietnam/Binh21/2015 lineage strains and 14

SD-06-2018 indicated that in SD-06-2018 the deletion region appears to have a higher degree of antigenic change than that of other strains, indicating it may be involved in PDCoV immune escape. S1 NTD has higher sequence variability (Figure 5). The N protein is the predominant antigen produced in coronavirus-infected cells, making it a major viral target (Kocherhans et al., 2001). The coronavirus N protein is a multifunctional protein involved in virus assembly, translation, apoptosis induction

ro of

and host innate immune defence. SARS-CoV N protein not only modulates the host

cell cycle by regulating cyclin-CDK activity but also inhibits the synthesis of type-1 interferon (1FN) (Chang et al., 2014; Liao et al., 2005). PDCoV N protein suppressed

-p

Sendai virus (SEV)-induced IFN-β production and transcription factor IRF3

re

activation (Chen et al., 2019a). Furthermore, the N-terminal region (1-246 aa) interacts with pRIG-I and interferes with its function (Chen et al., 2019a). The N

lP

protein analysed showed few differences among the three lineage strains. They shared

na

97.3%-99.3% sequence identity with the three lineage strains. Analysis showed that the N protein and S protein evolutionary rates are inconsistent.

ur

N protein is the general target for PDCoV detection, and the genetic extent of

Jo

variability should be evaluated carefully.

Conclusions In summary, this study provides more information about the deletion and genetic diversity of PDCoV. Phylogenetic analysis revealed that these strains belonged to the Chinese lineage. Meanwhile, new PDCoV strains with different patterns of deletions 15

in Nsp2 and Nsp3 are emerging, and these strains may lead to increased pathogenicity of the virus. Our study provides useful information to prevent PDCoV in China.

Conflict of Interest statement The authors declared no potential conflict of interest with respect to the research,

ro of

authorship, and/or publication of this article.

Acknowledgments

-p

This work was supported by the Wenzhou Basic Agricultural Science and

re

Technology Project (Grant Numbers N20180010 and N20190005).

lP

References

Alfuwaires, M., Altaher, A., Kandeel, M., 2017. Molecular Dynamic Studies of Interferon and Innate Immunity Resistance in MERS CoV Non-Structural Protein 3. Biological & pharmaceutical bulletin 40, 345-351.

na

Chang, C.K., Hou, M.H., Chang, C.F., Hsiao, C.D., Huang, T.H., 2014. The SARS coronavirus nucleocapsid protein--forms and functions. Antiviral research 103, 39-50. Chen, C.J., Makino, S., 2004. Murine coronavirus replication induces cell cycle arrest in G0/G1 phase.

ur

Journal of virology 78, 5658-5669. Chen, C.Y., Chang, C.K., Chang, Y.W., Sue, S.C., Bai, H.I., Riang, L., Hsiao, C.D., Huang, T.H., 2007. Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain

Jo

suggests a mechanism for helical packaging of viral RNA. Journal of molecular biology 368, 1075-1086.

Chen, J., Fang, P., Wang, M., Peng, Q., Ren, J., Wang, D., Peng, G., Fang, L., Xiao, S., Ding, Z., 2019a. Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-beta production by impairing dsRNA and PACT binding to RIG-I. Virus genes.

Chen, J., Fang, P., Wang, M., Peng, Q., Ren, J., Wang, D., Peng, G., Fang, L., Xiao, S., Ding, Z., 2019b. Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-beta production by impairing dsRNA and PACT binding to RIG-I. Virus genes 55, 520-531. Chen, P., Wang, K., Hou, Y., Li, H., Li, X., Yu, L., Jiang, Y., Gao, F., Tong, W., Yu, H., Yang, Z., Tong, G., Zhou, Y., 2019c. Genetic evolution analysis and pathogenicity assessment of porcine 16

epidemic diarrhea virus strains circulating in part of China during 2011-2017. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 69, 153-165. Chiou, H.Y., Huang, Y.L., Deng, M.C., Chang, C.Y., Jeng, C.R., Tsai, P.S., Yang, C., Pang, V.F., Chang, H.W., 2017. Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan. Transboundary and emerging diseases 64, 157-166. de Haan, C.A., Rottier, P.J., 2005. Molecular interactions in the assembly of coronaviruses. Advances in virus research 64, 165-230. Ding, Z., Fang, L., Jing, H., Zeng, S., Wang, D., Liu, L., Zhang, H., Luo, R., Chen, H., Xiao, S., 2014. Porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between IRF3 and TBK1. Journal of virology 88, 8936-8945. Dong, N., Fang, L., Yang, H., Liu, H., Du, T., Fang, P., Wang, D., Chen, H., Xiao, S., 2016. Isolation, CHN-HN-2014. Veterinary microbiology 196, 98-106.

ro of

genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain Dong, N., Fang, L., Zeng, S., Sun, Q., Chen, H., Xiao, S., 2015. Porcine Deltacoronavirus in Mainland China. Emerging infectious diseases 21, 2254-2255.

Fang, P., Fang, L., Hong, Y., Liu, X., Dong, N., Ma, P., Bi, J., Wang, D., Xiao, S., 2017. Discovery of a

novel accessory protein NS7a encoded by porcine deltacoronavirus. The Journal of general

-p

virology 98, 173-178.

Fang, P., Fang, L., Liu, X., Hong, Y., Wang, Y., Dong, N., Ma, P., Bi, J., Wang, D., Xiao, S., 2016. Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6.

re

Virology 499, 170-177.

Forni, D., Cagliani, R., Mozzi, A., Pozzoli, U., Al-Daghri, N., Clerici, M., Sironi, M., 2016. Extensive Positive Selection Drives the Evolution of Nonstructural Proteins in Lineage C

lP

Betacoronaviruses. Journal of virology 90, 3627-3639.

Fu, X., Fang, B., Liu, Y., Cai, M., Jun, J., Ma, J., Bu, D., Wang, L., Zhou, P., Wang, H., Zhang, G., 2018. Newly emerged porcine enteric alphacoronavirus in southern China: Identification, origin and evolutionary history analysis. Infection, genetics and evolution : journal of molecular

na

epidemiology and evolutionary genetics in infectious diseases 62, 179-187. Hsu, T.H., Liu, H.P., Chin, C.Y., Wang, C., Zhu, W.Z., Wu, B.L., Chang, Y.C., 2018. Detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in Taiwan. Archives of virology

ur

163, 3113-3117.

Hu, H., Jung, K., Vlasova, A.N., Chepngeno, J., Lu, Z., Wang, Q., Saif, L.J., 2015. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States.

Jo

Journal of clinical microbiology 53, 1537-1548.

Janetanakit, T., Lumyai, M., Bunpapong, N., Boonyapisitsopa, S., Chaiyawong, S., Nonthabenjawan, N., Kesdaengsakonwut, S., Amonsin, A., 2016. Porcine Deltacoronavirus, Thailand, 2015. Emerging infectious diseases 22, 757-759.

Jang, G., Lee, K.K., Kim, S.H., Lee, C., 2017. Prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in South Korea, 2014-2016. Transboundary and emerging diseases 64, 1364-1370. Le, V.P., Song, S., An, B.H., Park, G.N., Pham, N.T., Le, D.Q., Nguyen, V.T., Vu, T.T.H., Kim, K.S., Choe, S., An, D.J., 2018. A novel strain of porcine deltacoronavirus in Vietnam. Archives of virology 163, 203-207. 17

Lee, S., Lee, C., 2015. Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus. Virus research 208, 136-145. Li, D., Feng, H., Liu, Y., Chen, Y., Wei, Q., Wang, J., Liu, D., Huang, H., Su, Y., Wang, D., Cui, Y., Zhang, G., 2018a. Molecular evolution of porcine epidemic diarrhea virus and porcine deltacoronavirus strains in Central China. Research in veterinary science 120, 63-69. Li, W., Hulswit, R.J.G., Kenney, S.P., Widjaja, I., Jung, K., Alhamo, M.A., van Dieren, B., van Kuppeveld, F.J.M., Saif, L.J., Bosch, B.J., 2018b. Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility. Proceedings of the National Academy of Sciences of the United States of America 115, E5135-E5143. Li, Z.L., Zhu, L., Ma, J.Y., Zhou, Q.F., Song, Y.H., Sun, B.L., Chen, R.A., Xie, Q.M., Bee, Y.Z., 2012. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China. Virus genes 45, 181-185. Liang, Q., Li, B., Zhang, H., Hu, H., 2019. Complete Genome Sequences of Two Porcine

ro of

Deltacoronavirus Strains from Henan Province, China. Microbiology resource announcements 8.

Liao, Q.J., Ye, L.B., Timani, K.A., Zeng, Y.C., She, Y.L., Ye, L., Wu, Z.H., 2005. Activation of NF-kappaB by the full-length nucleocapsid protein of the SARS coronavirus. Acta biochimica et biophysica Sinica 37, 607-612.

-p

Likai, J., Shasha, L., Wenxian, Z., Jingjiao, M., Jianhe, S., Hengan, W., Yaxian, Y., 2019. Porcine

Deltacoronavirus Nucleocapsid Protein Suppressed IFN-beta Production by Interfering Porcine RIG-I dsRNA-Binding and K63-Linked Polyubiquitination. Frontiers in immunology 10,

re

1024.

Lin, C.M., Saif, L.J., Marthaler, D., Wang, Q., 2016. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains. Virus research 226, 20-39.

lP

Liu, B.J., Zuo, Y.Z., Gu, W.Y., Luo, S.X., Shi, Q.K., Hou, L.S., Zhong, F., Fan, J.H., 2018. Isolation and phylogenetic analysis of porcine deltacoronavirus from pigs with diarrhoea in Hebei province, China. Transboundary and emerging diseases 65, 874-882. Lorsirigool, A., Saeng-Chuto, K., Madapong, A., Temeeyasen, G., Tripipat, T., Kaewprommal, P.,

na

Tantituvanont, A., Piriyapongsa, J., Nilubol, D., 2017. The genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in Thailand in 2015. Virus genes 53, 240-248.

ur

Lorsirigool, A., Saeng-Chuto, K., Temeeyasen, G., Madapong, A., Tripipat, T., Wegner, M., Tuntituvanont, A., Intrakamhaeng, M., Nilubol, D., 2016. The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR. Archives of virology 161,

Jo

2909-2911.

Luo, J., Fang, L., Dong, N., Fang, P., Ding, Z., Wang, D., Chen, H., Xiao, S., 2016. Porcine deltacoronavirus (PDCoV) infection suppresses RIG-I-mediated interferon-beta production. Virology 495, 10-17.

Mai, K., Feng, J., Chen, G., Li, D., Zhou, L., Bai, Y., Wu, Q., Ma, J., 2018a. The detection and phylogenetic analysis of porcine deltacoronavirus from Guangdong Province in Southern China. Transboundary and emerging diseases 65, 166-173. Mai, K., Li, D., Wu, J., Wu, Z., Cheng, J., He, L., Tang, X., Zhou, Z., Sun, Y., Ma, J., 2018b. Complete Genome Sequences of Two Porcine Deltacoronavirus Strains, CHN-GD16-03 and CHN-GD16-05, Isolated in Southern China, 2016. Genome announcements 6. 18

McBride, R., van Zyl, M., Fielding, B.C., 2014. The coronavirus nucleocapsid is a multifunctional protein. Viruses 6, 2991-3018. Nelson, S.W., Zentkovich, M.M., Nolting, J.M., Bowman, A.S., 2019. Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus not Detected in Waterfowl in the North American Mississippi Migratory Bird Flyway in 2013. Journal of wildlife diseases 55, 223-226. Neuman, B.W., 2016. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles. Antiviral research 135, 97-107. Niederwerder, M.C., Hesse, R.A., 2018. Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada. Transboundary and emerging diseases 65, 660-675. Nogales, A., Marquez-Jurado, S., Galan, C., Enjuanes, L., Almazan, F., 2012. Transmissible gastroenteritis coronavirus RNA-dependent RNA polymerase and nonstructural proteins 2, 3, and 8 are incorporated into viral particles. Journal of virology 86, 1261-1266.

ro of

Perez-Rivera, C., Ramirez-Mendoza, H., Mendoza-Elvira, S., Segura-Velazquez, R., Sanchez-Betancourt, J.I., 2019. First report and phylogenetic analysis of porcine deltacoronavirus in Mexico. Transboundary and emerging diseases.

Phan, M.V.T., Ngo Tri, T., Hong Anh, P., Baker, S., Kellam, P., Cotten, M., 2018. Identification and characterization of Coronaviridae genomes from Vietnamese bats and rats based on

-p

conserved protein domains. Virus evolution 4, vey035.

Qing, Y., Liu, J., Huang, X., Li, Y., Zhang, Y., Chen, J., Wen, X., Cao, S., Wen, Y., Wu, R., Yan, Q., Ma, X., 2016. Immunogenicity of transmissible gastroenteritis virus (TGEV) M gene delivered by

re

attenuated Salmonella typhimurium in mice. Virus genes 52, 218-227.

Saeng-Chuto, K., Lorsirigool, A., Temeeyasen, G., Vui, D.T., Stott, C.J., Madapong, A., Tripipat, T., Wegner, M., Intrakamhaeng, M., Chongcharoen, W., Tantituvanont, A., Kaewprommal, P.,

lP

Piriyapongsa, J., Nilubol, D., 2017. Different Lineage of Porcine Deltacoronavirus in Thailand, Vietnam and Lao PDR in 2015. Transboundary and emerging diseases 64, 3-10. Shan, Y., Liu, Z.Q., Li, G.W., Chen, C., Luo, H., Liu, Y.J., Zhuo, X.H., Shi, X.F., Fang, W.H., Li, X.L., 2018. Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize

na

interferon-lambda production by blocking the nuclear factor-kappaB nuclear translocation. Journal of Zhejiang University. Science. B 19, 570-580. Shang, J., Zheng, Y., Yang, Y., Liu, C., Geng, Q., Tai, W., Du, L., Zhou, Y., Zhang, W., Li, F., 2018.

ur

Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State. Journal of virology 92.

Shi, D., Shi, H., Sun, D., Chen, J., Zhang, X., Wang, X., Zhang, J., Ji, Z., Liu, J., Cao, L., Zhu, X., Yuan, J.,

Jo

Dong, H., Wang, X., Chang, T., Liu, Y., Feng, L., 2017. Nucleocapsid Interacts with NPM1 and Protects it from Proteolytic Cleavage, Enhancing Cell Survival, and is Involved in PEDV Growth. Scientific reports 7, 39700.

Shokri, S., Mahmoudvand, S., Taherkhani, R., Farshadpour, F., 2019. Modulation of the immune response by Middle East respiratory syndrome coronavirus. Journal of cellular physiology 234, 2143-2151. Song, D., Zhou, X., Peng, Q., Chen, Y., Zhang, F., Huang, T., Zhang, T., Li, A., Huang, D., Wu, Q., He, H., Tang, Y., 2015. Newly Emerged Porcine Deltacoronavirus Associated With Diarrhoea in Swine in China: Identification, Prevalence and Full-Length Genome Sequence Analysis. Transboundary and emerging diseases 62, 575-580. 19

Su, M., Li, C., Guo, D., Wei, S., Wang, X., Geng, Y., Yao, S., Gao, J., Wang, E., Zhao, X., Wang, Z., Wang, J., Wu, R., Feng, L., Sun, D., 2016. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus. The Journal of veterinary medical science 78, 601-606. Suzuki, T., Shibahara, T., Imai, N., Yamamoto, T., Ohashi, S., 2018. Genetic characterization and pathogenicity of Japanese porcine deltacoronavirus. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 61, 176-182. Tan, Y.W., Fang, S., Fan, H., Lescar, J., Liu, D.X., 2006. Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells. Nucleic acids research 34, 4816-4825. Wang, L., Byrum, B., Zhang, Y., 2014. Porcine coronavirus HKU15 detected in 9 US states, 2014. Emerging infectious diseases 20, 1594-1595.

ro of

Wang, L., Su, S., Bi, Y., Wong, G., Gao, G.F., 2018a. Bat-Origin Coronaviruses Expand Their Host Range to Pigs. Trends in microbiology 26, 466-470.

Wang, M., Wang, Y., Baloch, A.R., Pan, Y., Tian, L., Xu, F., Shivaramu, S., Chen, S., Zeng, Q., 2018b. Detection and genetic characterization of porcine deltacoronavirus in Tibetan pigs

surrounding the Qinghai-Tibet Plateau of China. Transboundary and emerging diseases 65,

-p

363-369.

Wang, X.Y., Ji, C.J., Zhang, X., Xu, D.P., Zhang, D.L., 2018c. Infection, genetic and virulence characteristics of porcine epidemic diarrhea virus in northwest China. Infection, genetics and

re

evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 62, 34-39.

Wang, Y.W., Yue, H., Fang, W., Huang, Y.W., 2015. Complete Genome Sequence of Porcine announcements 3.

lP

Deltacoronavirus Strain CH/Sichuan/S27/2012 from Mainland China. Genome Woo, P.C., Huang, Y., Lau, S.K., Yuen, K.Y., 2010. Coronavirus genomics and bioinformatics analysis. Viruses 2, 1804-1820.

na

Woo, P.C., Lau, S.K., Lam, C.S., Lau, C.C., Tsang, A.K., Lau, J.H., Bai, R., Teng, J.L., Tsang, C.C., Wang, M., Zheng, B.J., Chan, K.H., Yuen, K.Y., 2012. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source

ur

of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. Journal of virology 86, 3995-4008.

Xia, L., Yang, Y., Wang, J., Jing, Y., Yang, Q., 2018. Impact of TGEV infection on the pig small intestine.

Jo

Virology journal 15, 102.

Xu, Z., Zhong, H., Zhou, Q., Du, Y., Chen, L., Zhang, Y., Xue, C., Cao, Y., 2018. A Highly Pathogenic Strain of Porcine Deltacoronavirus Caused Watery Diarrhea in Newborn Piglets. Virologica Sinica 33, 131-141.

Yuan, L., Chen, Z., Song, S., Wang, S., Tian, C., Xing, G., Chen, X., Xiao, Z.X., He, F., Zhang, L., 2015. p53 degradation by a coronavirus papain-like protease suppresses type I interferon signaling. The Journal of biological chemistry 290, 3172-3182. Zeng, S., Zhang, H., Ding, Z., Luo, R., An, K., Liu, L., Bi, J., Chen, H., Xiao, S., Fang, L., 2015. Proteome analysis of porcine epidemic diarrhea virus (PEDV)-infected Vero cells. Proteomics 15, 1819-1828. 20

Zhang, H., Liang, Q., Li, B., Cui, X., Wei, X., Ding, Q., Wang, Y., Hu, H., 2019a. Prevalence, phylogenetic and evolutionary analysis of porcine deltacoronavirus in Henan province, China. Preventive veterinary medicine 166, 8-15. Zhang, J., Chen, J., Shi, D., Shi, H., Zhang, X., Liu, J., Cao, L., Zhu, X., Liu, Y., Wang, X., Ji, Z., Feng, L., 2019b. Porcine deltacoronavirus enters cells via two pathways: A protease-mediated one at the cell surface and another facilitated by cathepsins in the endosome. The Journal of biological chemistry. Zhang, M.J., Liu, D.J., Liu, X.L., Ge, X.Y., Jongkaewwattana, A., He, Q.G., Luo, R., 2019c. Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China. Archives of virology 164, 413-425. Zhang, Q., Liu, X., Fang, Y., Zhou, P., Wang, Y., Zhang, Y., 2017. Detection and phylogenetic analyses of spike genes in porcine epidemic diarrhea virus strains circulating in China in 2016-2017. Virology journal 14, 194.

ro of

Zhang, X., Shi, H., Chen, J., Shi, D., Dong, H., Feng, L., 2015. Identification of the interaction between

vimentin and nucleocapsid protein of transmissible gastroenteritis virus. Virus research 200, 56-63.

Zhang, X., Shi, H., Chen, J., Shi, D., Li, C., Feng, L., 2014. EF1A interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication. Veterinary

-p

microbiology 172, 443-448.

Zhang, Y., Cheng, Y., Xing, G., Yu, J., Liao, A., Du, L., Lei, J., Lian, X., Zhou, J., Gu, J., 2019d. Detection and spike gene characterization in porcine deltacoronavirus in China during 2016-2018.

re

Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 73, 151-158.

Zhou, L., Sun, Y., Lan, T., Wu, R., Chen, J., Wu, Z., Xie, Q., Zhang, X., Ma, J., 2019. Retrospective

lP

detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern China. Transboundary and emerging diseases 66, 687-695. Zhou, P., Fan, H., Lan, T., Yang, X.L., Shi, W.F., Zhang, W., Zhu, Y., Zhang, Y.W., Xie, Q.M., Mani, S., Zheng, X.S., Li, B., Li, J.M., Guo, H., Pei, G.Q., An, X.P., Chen, J.W., Zhou, L., Mai, K.J., Wu, Z.X.,

na

Li, D., Anderson, D.E., Zhang, L.B., Li, S.Y., Mi, Z.Q., He, T.T., Cong, F., Guo, P.J., Huang, R., Luo, Y., Liu, X.L., Chen, J., Huang, Y., Sun, Q., Zhang, X.L., Wang, Y.Y., Xing, S.Z., Chen, Y.S., Sun, Y., Li, J., Daszak, P., Wang, L.F., Shi, Z.L., Tong, Y.G., Ma, J.Y., 2018. Fatal swine acute diarrhoea

ur

syndrome caused by an HKU2-related coronavirus of bat origin. Nature 556, 255-258.

Jo

Table 1 List of primers used in the study

Primer name

PDCoV S1-F PDCoV S1-R PDCoV S2-F PDCoV S2-F PDCoV N-F PDCoV N-R PDCoV Nsp2-3F PDCoV Nsp2-3R

Primer sequence (5’–3’)

Size (bp)

Target Genes

5’-ATGCAGAGAGCTCTATTGATTATGAC-3’ 5’-AACTTGCAAGTACTCCGTCTGAACG-3’ 5’-ATTTTCTCTTTCCGTTCAGACGGAG-3’ 5’-CTACCATTCCTTAAACTTAAAGGACG-3’ 5’-ATGGCTGCACCAGTAGTCCCTA-3’ 5’-CTACGCTGCTGATTCCTGCTTTAT-3’ 5’-TGGCCCGTCTAAACCTGGGGATGTCAT-3’ 5’-ATGGACCTCAGGATCCGGTACTGAGT-3’

1763 bp

S1

1750 bp

S2

1045 bp

N

1290 bp

ORF1a/1b

21

Table 2 PDCoV strains described in this study Strain

Accession No.

Collection date

Collection Country

Length (bp)

1 2 3 4 5 6 7 8 9 10 11 12

HKU15-44 USA/Iowa136/2015 USA/Nebraska145/2015 USA/Michigan448/2014 USA/Indiana453/2014 USA/Nebraska210/2014 OhioCVM1/2014 IL/2014/026PDV_P11 USA/Minnesota292/2014 YMG/JPN/2014 KNU14-04 P1_16_BTL_0115/PDCo V/2016/Lao Vietnam/Binh21/2015 Thailand/S5015L/2015 CH/SXD1/2015 CHN-JS-2014 CH/Sichuan/S27/2012 CHN-AH-2004 CHN-HB-2014 CHN/Tianjin/2016 CH/JXJGS01/2016 CH/Hunan/2014 CH/Jiangsu/2014 CHN-HG-2017 GD SD CHN/GS/2017/1 CHN-HN-1601 HNZK-02

JQ065042 KX022602 KX022605 KR265850 KR265851 KR265861 KJ769231.1 KP981395.1 KR265864 LC260044 KM820765 KX118627

2009 2015 2015 2014 2014 2014 2014 2014 2014 2014 2012 2016

China: Hong Kong USA: Iowa USA: Nebraska USA: Michigan USA: Indiana USA: Nebraska USA USA: Illinois USA: Minnesota Japan: Yamagata South Korea Laos

25430 25382 25320 25394 25394 25404 25433 25422 25395 25362 25422 25405

KX834352 KU051649 KT021234 KP757892 KT266822 KP757890 KP757891 KY065120 KY293677 KY513724 KY513725 MF095123 MF431742 MF431743 MF642324 MG832584 MH708123

2015 2015 2015 2014 2012 2004 2014 2016 2016 2014 2014 2017 2015 2014 2017 2016 2018

Viet Nam Thailand China: Shangxi China: Jangsu China: Sichuan China: Anhui China: Heibei China: Tianjin China: Jiangxi China: Hunan China: Jiangsu China China: Guangdong China: Shandong China: Gansu China China: Henan

25406 25405 25419 25420 25404 25420 25420 25413 25438 25413 25422 25399 25420 25414 25420 25419 25453

-p

re

lP

na

ur

Jo

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

ro of

No.

22

23

ro of

-p

re

lP

na

ur

Jo

ro of -p re

Jo

ur

na

lP

Fig.1. Phylogenetic analyses of PDCoV using the neighbour-joining algorithm and 1,000 bootstrap replications in a heuristic search with 29 PDCoV reference strains available in GenBank. (A) S protein genomic sequence. (B) N protein sequence. (C) Partial ORF1a gene.

24

ro of -p re lP na

Jo

ur

Fig.2. Analysis and comparison of amino acid mutations of the N protein of 10 PDCoV strains in Shandong, China

25

f oo pr ePr na l Jo ur

Fig.3. Analysis and comparison of amino acid mutations of the S protein of 10 PDCoV strains in Shandong, China.

26

ro of -p re

Jo

ur

na

lP

Fig.4. Amino acid sequence alignments of the partial ORF1gene at 369-791 aa. Two discontinuous amino acid deletions at positions 400-401 (yellow regions) and 758-760 (yellow regions) in Nsp2 and Nsp3 are also observed in SD01-2018, SD03-2018, SD05-2018, SD08-2018, the Vietnam/Laos/Thailand lineage and CH/Sichuan/S27/ 2012 strains. An additional 7 deletions at positions 755-761 (blue regions) in Nsp3 are observed in the SD07-2018, SD09-2018, SD10-2018 and SD11-2018 strains. Three continuous amino acid deletions at positions 758-760 (green regions) in Nsp3 are observed in SD12-2018 and CHN-HG-2017 strains. Two continuous amino acid deletions at positions 401-402 (black regions) in Nsp2 of the SD reference strain.

27

ro of -p

Jo

ur

na

lP

re

Fig.5. Schematic diagrams of the PDCoV genomic structure. (A) Organization of the PDCoV genome. (B) Organization of the S genome and locations of unique amino acids (aa). (C) Comparison of the antigenic index profiles of the S protein among the IL/2014/026PDV_P11 (representative USA/Japan/South Korea lineage strain), CHN-HG-2017 (representative China lineage strain), Vietnam/Binh21/2015 (representative Vietnam/Laos/Thailand lineage strain) and SD-06-2018. (D) Amino acid deletions/insertions are shown with IL/2014/026PDV_P11, CHN-HG-2017, Vietnam/Binh21/2015 and SD-06-2018 strains.

28

Supplementary

Table S1 PDCoV strains described in this study.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

S582N S579N HKU15-44 HKU15-155 IN2847 IL2768 SD3424 KY4813 PA3148 NE3579 OH1987 OH11846 MI6148 Michigan/8977/2014 Illinois121/2014 8734/USA-IA/2014, Illinois133/2014 Illinois134/2014 Illinois136/2014 Ohio137/2014 Arkansas61/2015 IL/2014/026PDV_P11 Minnesota442/2014 Minnesota214/2014 Michigan447/2014 Michigan448/2014 Indiana453/2014 Illinois449/2014 Minnesota/2013 Minnesota454/2014 Minnesota455/2014 Illinois272/2014 Illinois273/2014 NorthCarolina452/2014

LC216915.1 LC216914.1 JQ065042.2 NC_039208 KJ569769.1 KJ584355.1 KJ584356.1 KJ584357.1 KJ584358.1 KJ584359.1 KJ462462.1 KT381613.1 KJ620016.1 KM012168.1 KJ481931.1 KJ567050.1 KJ601777.1 KJ601778.1 KJ601779.1 KJ601780.1 KR150443.1 KP981395.1 KR265847.1 KR265848.1 KR265849.1 KR265850.1 KR265851.1 KR265852.1 KR265853.1 KR265854.1 KR265855.1 KR265856.1 KR265857.1 KR265858.1

Minnesota159/2014 Nebraska209/2014 Nebraska210/2014 Ohio444/2014

KR265859.1 KR265860.1 KR265861.1 KR265862.1

lP

na

ur

Jo 35 36 37 38

Collection date 2014 2014 2009 2010 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2004 2014 2014 2014 2015 2014 2014 2014 2014 2014 2014 2014 2013 2014 2014 2014 2014 2014

29

Collection Country

Length (bp)

China: Hong Kong China: Hong Kong China: Hong Kong China: Hong Kong USA: Ohio USA: Ohio USA: South Dakota USA: Kentucky USA: Pennsylvania USA: Nebraska USA: Ohio USA: Ohio USA: Michigan USA: Michigan USA USA: Iowa USA: Illinois USA: Illinois USA: Illinois USA: Ohio USA: Arkansas USA: Illinois USA: Minnesota USA: Minnesota USA: Michigan USA: Michigan USA: Indiana USA: Illinois USA: Minnesota USA: Minnesota USA: Minnesota USA: Illinois USA: Illinois USA: North Carolina USA: Minnesota USA: Nebraska USA: Nebraska USA: Ohio

25406 25422 25430 25425 25422 25422 25422 25422 25422 25422 25422 25422 25422 25411 25406 25422 25408 25404 25404 25404 25398 25422 25394 25396 25393 25394 25394 25394 25394 25394 25394 25399 25394 25394

ro of

Accession No.

-p

Strain

re

No.

2014 2014 2014 2014

25401 25396 25404 25394

na

ur

30

25394 25395 25394 25382 25394 25382 25320 25433 25420 25420 25320 25419 25404 25320 25370 25420 25438 25420 25420 25404 25413 25438 25438 25396 25403 25413 25422 25414 25399 25402 25420 25414 25420 25420 25423 25414 25419 25453 25454 25453 25419 25414 25402 25413

ro of

USA: Ohio USA: Minnesota USA: Iowa USA: Iowa USA: Minnesota USA: Nebraska USA: Nebraska USA China China: Anhui China China China: Sichuan China China China China: Jiangxi China China China China: Tianjin China China China: Guangdong China: Guangdong China: Hunan China: Jiangsu China China China: Guangdong China: Guangdong China: Shandong China: Gansu China: Gansu China China China China: Henan China: Henan China: Henan China: Guangdong China China: Sichuan China: Sichuan

-p

2014 2014 2014 2015 2015 2015 2015 2014 2014 2004 2014 2015 2012 2014 2014 2015 2015 2015 2015 2016 2016 2016 2016 2016 2016 2014 2014 2016 2017 2016 2015 2014 2016 2016 2017 2017 2016 2018 2018 2018 2016 2018 2017 2018

re

KR265863.1 KR265864.1 KR265865.1 KX022602.1 KX022603.1 KX022604.1 KX022605.1 KJ769231.1 KP757892.1 KP757890.1 KP757891.1 KT021234.1 KT266822.1 KT336560.1 KU665558.1 KU981059.1 KR131621.1 KU981061.1 KU981062.1 KX443143.2 KY065120.1 KY293677.1 KY293678.1 KY363867.1 KY363868.1 KY513724.1 KY513725.1 MF041982.1 MF095123.1 MF280390.1 MF431742.1 MF431743.1 MF642322.1 MF642323.1 MF948005.1 MG242062.1 MG832584.1 MH708123.1 MH708124.1 MH708125.1 MH715491.1 MK005882.1 MK330604.1 MK330605.1

lP

Ohio445/2014 Minnesota292/2014 Iowa459/2014 Iowa136/2015 Minnesota140/2015 Nebraska137/2015 Nebraska145/2015 OhioCVM1/2014 CHN-JS-2014 CHN-AH-2004 CHN-HB-2014 CH/SXD1/2015 CH/Sichuan/S27/2012 CHN-HN-2014 CHN-LYG-2014 NH CHJXNI2/2015 NH-P5 NH-P10 CH-01 CHN/Tianjin/2016 CH/JXJGS01/2016 CH/JXJGS02/2016 CHN-GD16-03 CHN-GD16-05 CH/Hunan/2014 CH/Jiangsu/2014 SHJS/SL/2016 CHN-HG-2017 CHN-GD-2016 GD SD CHN/GS/2016/1 CHN/GS/2016/2 HB-BD CHN-HeB1-2017 CHN-HN-1601 HNZK-02 HNZK-04 HNZK-06 CHGD/2016 CHN/SC/2018/1 CHN/Sichuan/2017 CHN/Sichuan/2018

Jo

39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

KY354363.1

2016

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

KNU14-04 KNU16-07-P5 KNU16-07 KNU16-07-P10 KNU16-11 AKT/JPN/2014 GNM-1/JPN/2014 GNM-2/JPN/2014 IWT/JPN/2014 MYZ/JPN/2014 OKN/JPN/2014 YMG/JPN/2014 HKD/JPN/2016 Vietnam/Binh21/2015 Vietnam/HaNoi6/2015 P1_16_BTL_0115/ PDCoV/2016/Lao TT_1115 Thailand/S5011/2015 Thailand/S5015L/2015

KM820765.1 MG837130.1 KY364365.1 MG837131.1 KY926512.1 LC260038.1 LC260039.1 LC260040.1 LC260041.1 LC260042.1 LC260043.1 LC260044.1 LC260045.1 KX834352.1 KX834351.1 KX118627.1

2014 2016 2014 2016 2016 2014 2014 2014 2014 2014 2014 2014 2016 2015 2015 2016 2015 2015 2015

re

KU984334.1 KU051641.1 KU051649.1

Jo

ur

na

lP

100 101 102

31

South Korea: Kyunggido South Korea South Korea South Korea South Korea South Korea Japan: Akita Japan: Gunma Japan: Gunma Japan: Iwate Japan: Miyazaki Japan: Okinawa Japan: Yamagata Japan: Hokkaido Viet Nam Viet Nam Laos

25422 25422 25422 25422 25422 25419 25362 25362 25362 25362 25362 25362 25362 25359 25406 25406 25405

ro of

DH1

-p

83

Thailand Thailand Thailand

25403 25405 25405